This invention is directed to an improved process for the synthesis of 2xe2x80x2-O-substituted pyrimidine nucleotides and oligomeric compounds containing these nucleotides. The invention features treating a 2,2xe2x80x2-anhydropyrimidine nucleoside or a 2S,2xe2x80x2-anhydropyrimidine nucleoside with a weak nucleophile and a Lewis acid. The process is economically advantageous relative to processes currently in use and is applicable to large scale synthesis. The invention further features oligomeric compounds having at least one modified pyrimidine monomeric sub-unit with modifications at 2xe2x80x2-O-position of the sugar and the 5 position of the pyrimidine. Oligomeric compounds of the invention exhibit increased binding affinity to nucleic acids and increased nuclease resistance. acids and increased nuclease resistance.
2xe2x80x2-O-Substituted pyrimidine nucleosides are useful per se in the preparation of oligonucleotides and related compounds. 2xe2x80x2-O-Substituted pyrimidine nucleosides are commercially available from companies such as, for example, Glen Research, Sterling, Va., and are considered to be items of commerce. The present invention is directed to new and useful processes for the preparation of 2xe2x80x2-O-substituted pyrimidine nucleosides.
Oligonucleotides and their analogs have been developed for various uses in molecular biology, including use as probes, primers, linkers, adapters, and gene fragments. Modifications to oligonucleotides used in these procedures include labeling with nonisotopic labels such as fluorescein, biotin, digoxigenin, alkaline phosphatase or other reporter molecules. Modifications also have been made to the ribose phosphate backbone to increase the nuclease stability of the resulting analog. These modifications include use of methyl phosphonates, phosphorothioates, phosphorodithioate linkages, and 2xe2x80x2-O-methyl ribose sugar units. Other modifications have been directed to the modulation of oligonucleotide uptake and cellular distribution. The success of these oligonucleotides for both diagnostic and therapeutic uses has created an ongoing demand for improved oligonucleotide analogs.
It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and man. For disease states, classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect may be obtained with minimal side effects. It is a general object of such therapeutic approaches to interfere with or otherwise modulate gene expression which would lead to undesired protein formation.
One method for inhibiting specific gene expression is with the use of oligonucleotides, especially oligonucleotides which are complementary to a specific target messenger RNA (mRNA) sequence. Several oligonucleotides are currently undergoing clinical trials for such use. Phosphorothioate oligonucleotides are presently being used as antisense agents in human clinical trials for various disease states, including use as antiviral agents.
Transcription factors interact with double-stranded DNA during regulation of transcription. Oligonucleotides can serve as competitive inhibitors of transcription factors to modulate the action of transcription factors. Several recent reports describe such interactions (see Bielinska, A., et. al., Science, 1990, 250, 997-1000; and Wu, H., et. al., Gene, 1990, 89, 203-209).
In addition to such use as both indirect and direct regulators of proteins, oligonucleotides have also found use in the diagnostic testing of materials including, for example, biological fluids, tissues, intact cells or isolated cellular components. As with gene expression inhibition, diagnostic applications utilize the ability of oligonucleotides to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligonucleotides via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
Oligonucleotides are also widely used as research reagents. They are particularly useful in studies exploring the function of biological molecules, as well as in the preparation of biological molecules. For example, the use of both natural and synthetic oligonucleotides as primers in PCR reactions has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies and genetic counseling. Commercialization has led to the development of kits which assist non-molecular biology-trained personnel in applying PCR.
Oligonucleotides are also used in other laboratory procedures. Several of these uses are described in common laboratory manuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and Current Protocols In Molecular Biology, F. M. Ausubel, et al., Eds., Current Publications, 1993. Representative of such uses are as Synthetic Oligonucleotide Probes, Screening Expression Libraries with Antibodies and Oligonucleotides, DNA Sequencing, In Vitro Amplification of DNA by the Polymerase Chain Reaction and Site-directed Mutagenesis of Cloned DNA (see Book 2 of Molecular Cloning, A Laboratory Manual, supra) and DNA-Protein Interactions and The Polymerase Chain Reaction (see Vol. 2 of Current Protocols In Molecular Biology, supra).
Oligonucleotides can be synthesized to have custom properties that are tailored for a desired use. Thus a number of chemical modifications have been introduced into oligonucleotides to increase their usefulness in diagnostics, as research reagents and as therapeutic entities. Such modifications include those designed to increase binding to a target strand (i.e. increase their melting temperatures, Tm), to assist in identification of the oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides, to provide a mode of disruption (a terminating event) once sequence-specifically bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide.
Gibson, K. J., and Benkovic, S. J., Nucleic Acids Research, 1987, 15, 6455-6467, report a phthalimide-protected 5-(3-aminopropyl)-2xe2x80x2-deoxyuridine nucleoside probe, which is incorporated into oligonucleotides.
Haralambidis, J., et.al., Nucleic Acids Research, 1987, 15, 4857-4876, reports C-5 substituted deoxyuridines which are incorporated into oligonucleotides. The substituent has a masked primary aliphatic amino group which can be further substituted with various groups.
PCT Application WO 94/17094, filed Jan. 22 1993, published Aug. 4, 1994, reports 5-substituted pyrimidine (cytosine or uracil) bases wherein the 5-substituent is C3-14 n-alkyl, C2-8 (E)-nxe2x88x921-alkenyl, ethynyl, or a C4-12 n-1-alkyl group, and the synthesis of oligonucleotides having one or more of the modified 5-substituted pyrimidine bases.
PCT Application No. WO 93/10820, filed Nov. 24, 1992, published Jun. 10, 1993, reports 5-(1-propynyl)uracil and 5-(1-propynyl)cytosine or related analogs, and the synthesis of oligonucleotides having one or more of the modified 5-substituted pyrimidine bases.
PCT Application No. WO 93/10820, filed Nov. 24, 1992, reports 2xe2x80x2- and 5-substituted pyrimidine nucleotides which are incorporated into oligonucleotides having a pi bond connecting the carbon atom attached to the 5xe2x80x2 position of the base.
The present invention provides oligomeric compounds having improved affinity for nucleic acid and having at least one monomeric sub-unit of structure I: 
wherein:
X is hydroxyl or amino;
R is halo or C1-C6 alkyl or substituted C1-C6 alkyl wherein said substitution is halo, amino, hydroxyl, thiol, ether or thioether;
L is oxygen or sulfur;
Z is fluoro or Oxe2x80x94R1X1, where R1 is C1-C6 alkyl, C6-C10 aryl, C7-C1-8 alkaryl and X1 is H, NH2 or imidazole; and
One of Q1 and Q2 is attached via a covalent bond to a nucleotide, oligonucleotide, nucleoside, or oligonucleoside and the other of said Q1 and Q2, is a hydroxyl, a protected hydroxyl, an activated solid support, a nucleotide, an oligonucleotide, a nucleoside, an oligonucleoside, an oligo-nucleotide/nucleoside, an activated phosphate, a phosphate, an activated phosphite, or a phosphite.
In one preferred embodiment of the invention, L is oxygen. In another embodiment Z is F.
In a further embodiment of the present invention oligomeric compounds are from about 5 to 200 sub-units in length. In a more preferred embodiment oligomeric compounds are from about 5 to 50 sub-units in length. In an even more preferred embodiment the oligomeric compounds are from about 10 to 20 sub-units in length.
In another embodiment, covalent bonds between monomeric sub-units of the invention and a nucleotide, oligonucleotide, nucleoside, or oligonucleoside in the oligomeric compound are chosen from phosphodiester, phosphotriester, hydrogen phosphonate, alkylphosphonate, alkylphosphonothioate, arylphosphonothioate, phosphorothioate, phosphorodithioate, or phosphoramidate.
In a further embodiment of the present invention oligomeric compounds are prepared having a plurality of monomeric sub-units of structure I. In a preferred embodiment, oligomeric compounds having a plurality of monomeric sub-units of structure I, are prepared having the monomeric sub-units located at preselected positions. Included in a particular embodiment of the invention is oligomeric compounds having at least one monomeric sub-unit of structure II: 
wherein:
X is hydroxyl or amino;
R is halo or C1-C6 alkyl or substituted C1-C6 alkyl wherein said substitution is halo, amino, hydroxyl, thiol, ether or thioether;
L is oxygen or sulfur; and
one of Q1 and Q2 is attached via a linking moiety to a nucleotide, oligonucleotide, nucleoside, or oligonucleoside and the other of said Q1 and Q2, is a hydroxyl, a protected hydroxyl, an activated solid support, a nucleotide, an oligonucleotide, a nucleoside, an oligonucleoside, an oligo-nucleotide/nucleoside, an activated phosphate, a phosphate, an activated phosphite, or a phosphite.
In a preferred embodiment of the invention L is O.
In a further embodiment, oligomeric compounds of the present invention are from about 5 to 50 sub-units in length.
In another embodiment, covalent bonds between monomeric sub-units of the invention and a nucleotide, oligonucleotide, nucleoside, or oligonucleoside in the oligomeric compound are chosen from phosphodiester, phospho-triester, hydrogen phosphonate, alkylphosphonate, alkylphosphonothioate, arylphosphonothioate, phosphorothioate, phosphorodithioate, or phosphoramidate.
In a further embodiment of the present invention oligomeric compounds are prepared having a plurality of monomeric sub-units of structure I. In a preferred embodiment, oligomeric compounds having a plurality of monomeric sub-units of structure I, are prepared having the monomeric sub-units located at preselected positions. Included in a particular embodiment of the invention is oligomeric compounds having at least one monomeric sub-unit of structure III: 
wherein:
X is hydroxyl or amino;
R is halo or C1-C6 alkyl or substituted C1-C6 alkyl wherein said substitution is halo, amino, hydroxyl, thiol, ether or thioether;
L is oxygen or sulfur;
R1 is C1-C6 alkyl, C6-C10 aryl, C7-C18 alkaryl and X1 is H, NH2 or imidazole; and
one of Q1 and Q2 is attached via a linking moiety to a nucleotide, oligonucleotide, nucleoside, or oligonucleoside and the other of said Q1 and Q2, is a hydroxyl, a protected hydroxyl, an activated solid support, a nucleotide, an oligonucleotide, a nucleoside, an oligonucleoside, an oligonucleotide/nucleoside, an activated phosphate, a phosphate, an activated phosphite, or a phosphite.
In a preferred embodiment of the invention, L is O.
In a further embodiment, oligomeric compounds of the present invention are from about 5 to 50 sub-units in length.
In another embodiment, covalent bonds between monomeric sub-units of the invention and a nucleotide, oligonucleotide, nucleoside, or oligonucleoside in the oligomeric compound are chosen from phosphodiester, phosphotriester, hydrogen phosphonate, alkylphosphonate, alkylphosphonothioate, arylphosphonothioate, phosphorothioate, phosphorodithioate, or phosphoramidate.
In a further embodiment of the present invention oligomeric compounds are prepared having a plurality of monomeric sub-units of structure I. In a preferred embodiment, oligomeric compounds having a plurality of monomeric sub-units of structure I, are prepared having the monomeric sub-units located at preselected positions.
Also in accordance with this invention there are provided improved processes for the synthesis of 2xe2x80x2-O-susbstituted pyrimidine nucleosides of formula: 
wherein:
Q is a pyrimidine base or a 2-S pyrimidine base;
R1 is substituted or unsubstituted C1-C30 alkyl, C1-C30 alkenyl, C1-C30 alkynyl, C6-C14 aryl, or C7-C30 aralkyl, wherein said substitution is halo, amino, hydroxyl, thiol, ether or thioether; and
R2 and R3 are independently hydrogen or a hydroxyl protecting group;
comprising the steps of:
providing a 2-2xe2x80x2-anhydropyrimidine nucleoside;
selecting an alcohol of the formula R1xe2x80x94OH; and
treating said 2-2xe2x80x2-anhydropyrimidine nucleoside and said alcohol with a Lewis acid under conditions of time, temperature and pressure effective to yield said 2xe2x80x2-O-substituted pyrimidine nucleoside.
In accordance with this invention there are also provided improved processes for the synthesis of a 2xe2x80x2-O-substituted cytidine nucleoside of formula: 
wherein:
Xxe2x80x2 is O or S;
R1 is substituted or unsubstituted C1-C30 alkyl, C1-C30 alkenyl, C1-C30 alkynyl, C6-C14 aryl, or C7-C30 aralkyl, wherein said substitution is halo, amino, hydroxyl, thiol, ether or thioether;
R2 and R3 are independently hydrogen or a hydroxyl protecting group;
R5 and R6 are independently H, C1-C30 hydrocarbyl or substituted C1-C30 hydrocarbyl;
comprising the steps of:
providing a 2-2xe2x80x2-anhydrouridine nucleoside of formula: 
selecting an alcohol of formula R1xe2x80x94OH;
treating said 2-2xe2x80x2-anhydrouridine nucleoside and said alcohol with a Lewis acid under conditions of time, temperature and pressure effective to form a 2xe2x80x2-O-substituted uridine nucleoside; and
aminating said 2xe2x80x2-O-substituted uridine nucleoside to said 2xe2x80x2-O-substituted cytidine nucleoside.
In preferred embodiments of the invention the 2-2xe2x80x2-anhydropyrimidine nucleoside and the alcohol of the formula
R1xe2x80x94OH are treated in a pressure sealed vessel as the reaction vessel, for example a pressure bomb. Preferably, the reaction vessel is heated from about 120xc2x0 C. to about 200xc2x0 C.
In a further embodiment of the invention the Lewis acid is a borate, particularly a trialkyl borate.
Preferably, the alkyl groups of the trialkyl borate are the same as the R group of the alcohol, so the formula of the borate is B(OR1)3. The trialkyl borate is preferably prepared from the treatment of borane with an alcohol.
Preferably, the alcohol that is used for the treatment of the 2,2xe2x80x2-anhydropyrimidine nucleoside is also used to prepare the trialkyl borate, preferably an alcohol of formula HOxe2x80x94R1.
In some preferred embodiments of the invention R1 is C1-C30 alkyl, more preferably C1-C10 alkyl. In another preferred embodiment the R1 is C6-C14 aryl.
In one preferred embodiment of the invention the pyrimidine nucleoside prepared by the process is uridine or 5-methyluridine.
In another preferred embodiment of the invention the 2xe2x80x2-O-substituted cytidine nucleoside prepared by the process is uridine or 2xe2x80x2-O-methyl-5-methylcytidine.
In accordance with this invention there are provided oligomeric compounds comprising at least one monomeric sub-unit of Structure IV: 
wherein:
A is hydroxyl or amino;
R is halo or C1-C6 alkyl or substituted C1-C6 alkyl wherein said substitution is halo, amino, hydroxyl, thiol, ether or thioether;
L is oxygen or sulfur;
Zxe2x80x2 is substituted or unsubstituted C1-C30 alkyl, C1-C30 alkenyl, C1-C30 alkynyl, C6-C14 aryl, or C7-C30 aralkyl, wherein said substitution is halo, amino, hydroxyl, thiol, ether or thioether; and
one of Q1 and Q2 is attached via a linking moiety to a nucleotide, oligonucleotide, nucleoside, or oligonucleoside and the other of said Q1 and Q2, is a hydroxyl, a protected hydroxyl, an activated solid support, a nucleotide, an oligonucleotide, a nucleoside, an oligonucleoside, an oligo-nucleotide/nucleoside, an activated phosphate, a phosphate, an activated phosphite, or a phosphite.
In preferred embodiments of the invention oligomeric compounds are provided having at least one monomeric sub-unit wherein L is O. In a further preferred embodiment oligomeric compounds are provided having at least one monomeric sub-unit wherein Zxe2x80x2 is substituted alkyl. In a more preferred embodiment Zxe2x80x2 is methoxyethyl (CH3OCH2CH2xe2x80x94).
In one embodiment the oligomeric compounds of the invention comprise from 5 to 200 monomeric sub-units. In a more preferred embodiment the oligomeric compounds of the invention comprise from 5 to 50 monomeric sub-units. In an even more preferred embodiment oligomeric compounds of the invention comprise from 10 to 20 sub-units.
In another embodiment one of Q1 and Q2 is attached via a linking moiety to a nucleotide, oligonucleotide, nucleoside, or oligonucleoside wherein the linking moiety comprises a phosphodiester, phosphotriester, hydrogen phosphonate, alkylphosphonate, alkylphosphonothioate, arylphosphonothioate, phosphorothioate, phosphorodithioate, or phosphoramidate.
In a further embodiment of the invention the oligomeric compounds are provided having a plurality of monomeric sub-units of the above formula. In a more preferred embodiment the oligomeric compounds are provided with the monomeric sub-units located at preselected positions.
Preferred oligomeric compounds of the invention have at least one monomeric sub-unit of Structure I. Structure I is a xcex2-D-erythro-pentofuranosyl sugar substituted at the 2xe2x80x2 position and coupled 1xe2x80x2 to 1 to a 5-substituted pyrimidine through a glycosyl linkage. The 5xe2x80x2 and 3xe2x80x2 ends of the monomeric sub-unit can be coupled to a nucleotide, nucleoside, oligonucleotide, oligonucleoside, or a mixed oligo-nucleotide/nucleoside or can be a 3xe2x80x2 or a 5xe2x80x2 terminal end of the oligomeric compound.
Monomeric sub-units used to prepare compounds of the invention include nucleotides and nucleosides. Nucleotides include a phosphorous linking moiety whereas nucleosides have a non phosphorous linking moiety and each have a ribofuranose moiety attached through a glycosyl bond to a nucleobase.
Monomeric sub-units of the invention are coupled using linking moieties. Linking moieties include phosphodiester, phosphotriester, hydrogen phosphonate, alkylphosphonate, alkylphosphonothioate, arylphosphonothioate, phosphorothioate, phosphorodithioate, phosphoramidate, ketone, sulfone, carbonate and thioamidate. Alkylphosphonothioate linkages are disclosed in WO 94/02499. Other such moieties can also be employed.
In one aspect of the present invention 2xe2x80x2-F-5-alkyl-uridine (and the 5-halo analog) monomeric sub-units are prepared by first substituting the appropriate alkyl group on the 5-position of the nucleoside. In the case of a 5-halo group, 5-F, Cl, Br, and I uracils are available through Aldrich Chemical Company. Substitution of alkyl, alkenyl, and alkynyl groups at C-5 of uracil is disclosed in PCT application PCT/US92/10115, filed Nov. 24, 1992, and examples of alkyl substitutions are further disclosed by Manoharan, M., Antisense Research and Applications, Crooke and Lebleu, eds., CRC Press, Boca Raton, 1993.
5-Alkylated uridine is converted into the 2,2xe2x80x2-anhydro[1-(xcex2-D-arabinofuranosyl)-5-alkyluridine] by treatment with diphenylcarbonate and sodium bicarbonate in DMF followed by purification. The 2,2xe2x80x2-anhydro[1-(xcex2-D-arabinofuranosyl)-5-alkyluridine] is further treated with HF/pyridine in an appropriate solvent, e.g. dioxane, to give 1-(2-fluoro-xcex2-D-erythro-pentofuranosyl)-5-alkyluridine. This compound is converted into the DMT/amidite following standard methods and techniques to give 1-(5-O-dimethoxytrityl-2-fluoro-3-Oxe2x80x94N,N-diisopropylamino-2-cyano-ethylphosphite-xcex2-D-erythro-pentofuranosyl)-5-alkyluridine. The 1-(5-O-dimethoxytrityl-2-fluoro-3-Oxe2x80x94N,N-diisopropyl-amino-2-cyanoethylphosphite-xcex2-D-erythro-pentofuranosyl)-5-alkyluridine is used as a monomeric sub-unit precursor in oligomeric compound synthesis.
Conversion of the 5-alkylated-2xe2x80x2-F-uridine to a 5-alkylated-2xe2x80x2-F-4-N-protected (e.g. benzoyl) cytidine is accomplished by known methods and techniques using 1,2,4-triazole. The 5-O-alkylated uridine is first protected at the 3xe2x80x2 and 5xe2x80x2 positions. This protection can be effected using acetic anhydride. 1,2,4-Triazole in an appropriate solvent (e.g. acetonitrile) with a base present (e.g. triethylamine) is treated with POCl3 at low temperature. The protected 5-O-alkylated-2xe2x80x2-F-uridine is dissolved in an appropriate solvent and added to the solution containing the triazole/POCl3. After sufficient time has passed and subsequent workup and purification the triazine-1-(3xe2x80x2,5xe2x80x2-di-O-acetyl-2-fluoro-xcex2-D-erythro-pentofuranosyl)-5-alkyluridine is obtained. This compound is converted into 5-alkyl-1-(2-fluoro-xcex2-D-erythro-pentofuranosyl)-Cytosine by treatment with ammonia. The exocycloamino group is protected for example by treatment with benzoic anhydride in a suitable solvent e.g. pyridine.
This compound is converted into the DMT/amidite following standard methods and techniques to give 4-N-protected-5-alkyl-1-(2-fluoro-3-Oxe2x80x94N,N-diisopropylamino-2-cyanoethylphosphite-5-O-dimethoxytrityl-xcex2-D-erythro-pentofuranosyl)-Cytosine. The 4-N-protected-5-alkyl-1-(2-fluoro-3-Oxe2x80x94N,N-diisopropylamino-2-cyanoethylphosphite-5-O-dimethoxytrityl-xcex2-D-erythro-pentofuranosyl)-Cytosine is used as a monomeric sub-unit precursor in oligomeric compound synthesis.
The preparation of 5-substituted-2xe2x80x2-F-pyrimidines using more complicated groups than alkyl (e.g. halo or substituted C1-C6 alkyl, wherein said substitutions are halo, amino, hydroxyl, thiol, ether or thioether) for the substituent of the 5-position can require that the group be protected prior to preparing the anhydro compound using an appropriate protecting group. The overall synthesis of compounds with protected groups at the 5 position is identical to that described above for incorporation of one of these substituted alkyl groups in place of a saturated alkyl group.
In another aspect of the present invention 2xe2x80x2-O-substituted-5-substituted uridine monomeric sub-units are prepared using the 2, 2xe2x80x2-anhydro[1-(xcex2-D-arabinofuranosyl)-5-alkyluridine procedures described except that to open the anhydro an alcohol is used e.g. phenol for phenyl substituent, or propanol for an O-propyl substituent.
The resulting compound is converted into the DMT/amidite following standard methods and techniques to give 1-(2-O-substituted-3-Oxe2x80x94N,N-diisopropylamino-2-cyanoethylphosphite-5-O-dimethoxytrityl-xcex2-D-erythro-pentofuranosyl)-5-substituted uridine. The DMT/amidite is used as a monomeric sub-unit precursor in oligomeric compound synthesis.
1-(2-O-substituted-5-dimethoxytrityl-xcex2-D-erythro-pentofuranosyl)-5-substituted uridine is converted into the cytosine analog by known methods and techniques using 1,2,4-triazole. The 5-substituted-2xe2x80x2-O-substituted uridine is first protected at the 3xe2x80x2 position using an appropriate protecting group e.g.acetic anhydride. The material is purified after workup. 1,2,4-Triazole in an appropriate solvent (e.g. acetonitrile) with a base present (e.g. triethylamine) is treated with POCl3 at low temperature. The protected 5-substituted-2xe2x80x2-O-substituted-3xe2x80x2-O-protected uridine is dissolved in an appropriate solvent and added to the solution containing the triazole/POCl3. After sufficient time has passed and subsequent workup and purification the 1-(2-O-substituted-3-O-acetyl-5-O-dimethoxytrityl-xcex2-D-erythro-pentofuranosyl)-4-triazolo-5-substituted pyrimidine is obtained which is converted into the cytidine analog by treatment with ammonia. The exocycloamino group (N-4) is protected for example by treatment with benzoic anhydride in a suitable solvent like pyridine or DMF and further converted into the DMT/amidite as illustrated above. The resulting 1-(2-O-substituted-3-Oxe2x80x94N,N-diisopropylamino-2-cyanoethylphosphite-5-O-dimethoxytrityl-xcex2-D-erythro-pento-furanosyl)-4-N-benzoyl-5-substituted cytidine is used as a monomeric sub-unit precursor in oligomeric compound synthesis.
In other embodiments of the invention the 2-S analogs of the 2xe2x80x2-substituted-5-substituted pyrimidines are prepared. The 2xe2x80x2-O-substituted-2-thio-5-substituted uridine is prepared in one method by starting with a 2,3,5-tri-O-benzoyl ribose sugar and coupling to a 2-thio-5-substituted pyrimidine via a glycosylation step. The synthesis of varied 2-thio-5-substituted pyrimidines are disclosed in Vorbruggen, P., et.al., Angew. Chem. Int. Ed., 1969, 8, 976-977, and in Vorbruggen, P., et.al., Chem. Ber., 1973, 106, 3039-3061. The 2,3,5-tri-O-benzoyl ribose sugar and a-5-substituted-2-thiouracil are dissolved in a suitable solvent and treated with Nxe2x80x94O-Bis(trimethyl silyl)acetamide and trimethyl silyl triflate. The resulting 2,3,5-tri-O-benzoyl-2-thio-5-substituted uridine is deprotected using sodium methoxide in an appropriate solvent to give 2-thio-5-substituted uridine. The 2-thio-5-substituted uridine is dissolved in a suitable solvent and treated with dibutyltin oxide and tetrabutyl ammonium iodide followed by an alkyl halide e.g. methyl iodide, to give the 2xe2x80x2-O-substituted-2-thio-5-substituted uridine
The 2xe2x80x2-O-substituted-2-thio-5-substituted uridine is converted into the DMT/amidite following standard methods and techniques to give 1-(2-O-substituted-3-Oxe2x80x94N,N-diisopropylamino-2-cyanoethylphosphite-5-O-dimethoxytrityl-xcex2-D-erythro-pentofuranosyl)-2-thio-5-substituted uridine. The DMT/amidite is used as a monomeric sub-unit precursor in oligomeric compound synthesis.
In still other embodiments of the invention the 2-S analogs of the 2xe2x80x2-F-5-substituted pyrimidines are prepared. One method of preparing 2xe2x80x2-F-2-Thio-5-substituted pyrimidines is to use a similar method to that used in the preparation of the 2xe2x80x2-F-2-substituted-5-substituted-pyrimidine. This involves doing selective protection of functional groups and forming the anhydro bond between S-2 and 2xe2x80x2, analogous to the anhydro formed above between the 0-2 and the 2xe2x80x2.
2,3,5-Tri-O-benzoyl-2-thio-5-methyl-uridine is formed via a glycosylation between 2,3,5-tri-O-benzoyl ribose and a 5-substituted-2-thiouridine. After deprotection with sodium methoxide in an appropriate solvent and purification the resulting 5-methyl-2-thiouridine is protected with DMT at the 5xe2x80x2-position using standard methods and techniques. Next, the 2xe2x80x2-position is protected with a t-butyl-dimethylsilyl group by treatment with t-butyldimethylsilyl chloride in an appropriate solvent.
The resulting 5xe2x80x2-O-dimethoxytrityl-3xe2x80x2-t-butyl-dimethylsilyl-5-substituted-2-thiouridine is dissolved in an appropriate solvent and treated with methanesulfonyl chloride to give the 5xe2x80x2-O-dimethoxytrityl-3xe2x80x2-t-butyl-dimethylsilyl-2xe2x80x2-methanesulfonyl-5-substituted-2-thiouridine. The 5xe2x80x2-O-dimethoxytrityl-3xe2x80x2-t-butyl-dimethylsilyl-2xe2x80x2-methanesulfonyl-5-substituted-2-thiouridine is further treated with sodium methoxide in an appropriate solvent to give the 5xe2x80x2-O-dimethoxytrityl-3xe2x80x2-t-butyl-dimethylsilyl-2-2xe2x80x2-thio anhydro-5-substituted-2-thiouridine. This compound is further treated with HF/pyridine in dioxane to give the 2xe2x80x2-Fluoro-3xe2x80x2-t-butyl-dimethylsilyl-5xe2x80x2-O-dimethoxytrityl-5-substituted-2-thiouridine.
The 2xe2x80x2-Fluoro-3xe2x80x2-t-butyl-dimethylsilyl-5xe2x80x2-O-dimethoxytrityl-5-substituted-2-thiouridine is converted into the amidite using standard methods and techniques to give 1-(2-Fluoro-3-Oxe2x80x94N,N-diisopropylamino-2-cyanoethylphosphite-5-O-dimethoxytrityl-xcex2-D-erythro-pentofuranosyl)-2-thio-5-substituted uridine. The DMT/amidite is used as a monomeric sub-unit precursor in oligomeric compound synthesis.
The conversion of the 2xe2x80x2-Fluoro-3xe2x80x2-t-butyl-dimethylsilyl-5xe2x80x2-O-dimethoxytrityl-5-substituted-2-thiouridine into the cytidine analog is accomplished using the above procedures for conversion of the 1-(2-O-substituted-5-dimethoxytrityl-xcex2-D-erythro-pentofuranosyl)-5-substituted uridine into its cytidine analog using the 1,2,4-triazole procedure. For the conversion of a 2-S compound this group must also be protected using an appropriate protecting group e.g. toluoyl.
In United States patent application entitled xe2x80x9cOligonucleotide Modulation of protein kinase C,xe2x80x9d Ser. No. 08/089,996, filed Jul. 9, 1995xe2x80x94also identified as attorney docket number ISIS-1154, commonly assigned with this application, the disclosure of which is herein incorporated by reference, gapped oligonucleotides (see, Dean, et.al., J. Biol. Chem., 1994, 23, 16416) were synthesized and tested in a protein kinase C assay to determine their potency. One exemplary oligonucleotide having good potency is oligonucleotide SEQ ID NO: 27, which is a 20 mer deoxyphosphorothioate. This oligonucleotide gave approximately 80% reduction of the smaller transcript and over 90% reduction of the larger transcript in the above described assay.
In one embodiment of the present invention oligomeric compounds having at least one monomeric unit of structure I are synthesized and used to inhibit the synthesis of the PKC-xcex1 protein. Two oligonucleotides having similar sequences to SEQ ID NO: 27 were synthesized as oligonucleotide SEQ IN NO: 28, which is a fully modified phosphorothioate having 2xe2x80x2-fluoro at positions 1-6 and 15-20, and uracils in place of the thymines at positions 2, 3, 5, and 16-18, and as oligonucleotide SEQ IN NO: 29, which is a fully modified phosphorothioate having 2xe2x80x2-fluoro-5-methyluridine in place of the thymidines at positions 2, 3, 5, and 16-18, and further having 2xe2x80x2-fluoros at positions 1, 4, 6, 15, 19, and 20. These two oligonucleotides (SEQ IN NO: 28, SEQ IN NO: 29) were evaluated in the above assay and the results compared with that of SEQ IN NO: 27.
SEQ IN NO: 28 and SEQ IN NO: 29 exhibited about a 10 fold increase in potency relative to SEQ IN NO: 27. SEQ IN NO: 29 also showed a measurable increase in potency relative to SEQ IN NO: 28.
As expected from the above results the Tm""s of these 3 oligonucleotides are in the order SEQ ID NO: 29 greater than SEQ ID NO: 28 greater than SEQ ID NO: 27.
In one aspect of the present invention the oligomeric compounds of the invention have a plurality of monomeric sub-units of Structure IV. 
Where the variables A, R, L, Q1, Q2, and Z are as described above.
In a further aspect of the present invention the ligomeric compounds of the invention having a plurality of onomeric sub-units of Structures I-IV, have a predetermined sequence. Monomeric sub-units of the invention can be located in predetermined positions in an oligomeric compound of predetermined sequence to increase the activity of the oligomeric compound.
In one aspect of the invention nucleoside dimers are incorporated into compounds of the invention. One procedure for incorporating a mixed oligo-nucleotide/nucleoside into a compound of the invention is to incorporate nucleotides, nucleoside dimers, and monomeric sub-units of Structures I-IV into oligomeric compounds in a predetermined order using standard oligonucleotide synthesis protocols. In one aspect of the invention nucleosides, oligonucleoside, and nucleoside dimers are prepared as per the disclosures of U.S. patent application Ser. No. 08/174,379, filed Dec. 28, 1993, entitled xe2x80x9cHydrazine-Based and Hydroxylamine-Based Oligonucleoside-Linkages and Bidirectional Synthetic Processes Thereforxe2x80x9d, also identified by attorney docket ISIS-0716; U.S. patent application Ser. No. 08/040,933, filed Mar. 31, 1993, entitled xe2x80x9cBackbone Modified Oligonucleotide Analogs And Preparation Thereof Radical Couplingxe2x80x9d, also identified by attorney docket ISIS-0717; and U.S. patent application Ser. No. 08/040,903, filed Mar. 31, 1993, entitled xe2x80x9cBackbone Modified Oligonucleotide Analogs And Preparation Thereof Through Reductive Couplingxe2x80x9d, also identified by attorney docket ISIS-0718, commonly assigned with this application, the disclosures of which are herein incorporated by reference.
An oligo-nucleotide/nucleoside for the purposes of the present invention is a mixed backbone oligomer having at least two nucleosides covalently bound by a non-phosphate linkage and forming a phosphorous containing covalent bond with a monomeric sub-unit as defined above. An oligonucleotide/nucleoside can have a plurality of nucleotides and nucleosides coupled through phosphorous containing and non-phosphorous containing linkages.
The processes of the present invention are useful for the synthesis of 2xe2x80x2-O-substituted pyrimidine nucleosides.
2xe2x80x2-O-substituted pyrimidine nucleosides are important compounds used routinely for the synthesis of oligonucleotides and related compounds. Representative commercially available 2xe2x80x2-O-substituted pyrimidine nucleosides include 2xe2x80x2-O-methyl uridine and 2xe2x80x2-O-methyl cytidine.
For the purposes of the present invention, pyrimidine nucleosides include naturally occurring pyrimidines bases such as uridine and cytidine as well as non-naturally occurring pyrimidines such as 2S analogs and 5- and 6-substituted uridines and cytidines. Also included are N3 substituted pyrimidines and pyrimidines having heterocyclic ring structures attached, for example, at position 4 and/or 5. Many modified pyrimidines amenable to the present invention are known in the art (See for example Chemistry of Nucleosides and Nucleotides, Volume 1 Plenum Press, N.Y. 1988). As defined herein, a 2S pyrimidine base is a pyrimidine base having a sulfur atom bound to the 2-position thereof.
Nucleobases according to the invention include purines and pyrimidines such as adenine, guanine, cytosine, uridine, and thymine, as well as other synthetic and natural nucleobases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halo uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo uracil), 4-thiouracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other B-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine. Further purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley and Sons, 1990, and those disclosed by Englisch, et al., Angewandte Chemie, International Edition 1991, 30, 613.
As defined herein, 2S,2xe2x80x2-anhydropyrimidine nucleosides are pyrimidine nucleosides which have a single bond connecting the 2-position of the pyrimidine ring to the 2xe2x80x2-oxygen of the nucleosidic sugar. 2S,2xe2x80x2-anhydropyrimidine nucleosides, as defined herein, are pyrimidine nucleosides wherein the 2-position of the pyrimidine ring is bound to the 2xe2x80x2-position of the nucleosidic sugar thorough an intervening sulfur atom.
In some preferred embodiments 2S,2xe2x80x2-anhydropyrimidine nucleosides or 2S,2xe2x80x2-anhydropyrimidine nucleosides have the general formula: 
wherein:
X is O or S;
R2 and R3 are independently hydrogen or a hydroxyl protecting group; and
R5 and R6 are independently H, C1-C30 hydrocarbyl or substituted C1-C30 hydrocarbyl;
2S,2xe2x80x2-Anhydropyrimidine nucleosides and 2S,2xe2x80x2-anhydropyrimidine nucleosides suitable for use in the processes of the invention include those that are unsubstituted as well as those having substitutions on the pyrimidine ring. A variety of such substituted pyrimidine substitutions are known in the art, for example, alkylation at the 5-position. See for example Chemistry of Nucleosides and Nucleotides, Volume 1 supra. Such substituted 2,2xe2x80x2 and 2S,2xe2x80x2-anhydropyrimidine nucleosides can be prepared from a naturally occurring nucloeside which can be modified before or after formation of the 2,2xe2x80x2 or 2S,2xe2x80x2 bond. Thus, in one aspect of the present invention, substituents are attached to the 2,2xe2x80x2 or 2S,2xe2x80x2-anhydropyrimidine nucleoside after formation of the 2,2xe2x80x2 or 2S,2xe2x80x2 bond, and in another aspect of the invention the attachment of substituents to the pyrimidine ring of the pyrimidine nucleoside precedes formation of the 2,2xe2x80x2 or 2S,2xe2x80x2-anhydropyrimidine nucleoside.
The process of the present invention provides significant economic benefits in the production of 2xe2x80x2-O-substituted pyrimidines. For example, the compound 2xe2x80x2-o-methyl uridine, which is an important intermediate in the synthesis of modified oligonucleotides and related compounds, can be prepared at about one tenth the cost of the commercially available material using the process of the present invention. Another useful intermediate, 2xe2x80x2-O-methyl-cytidine, also useful in the synthesis of modified oligonucleotides and related compounds, can be prepared at about one fifth the cost of the commercially available material using the process of the present invention. These costs are based on yields previously obtained for large scale synthesis, the price of the starting materials, and the hours of labor required.
2,2xe2x80x2-Anhydropyrimidine nucleosides can be prepared from pyrimidine nucleosides by treatment with diphenyl-carbonate and sodium bicarbonate in DMF, or other traditional solvents, followed by purification. See e.g., Townsend, Leroy B., Chemistry of Nucleosides and Nucleotides 1, Plenum Press, New York, 1988). The resulting 2,2xe2x80x2-anhydropyrimidine nucleosides are treated with a weak nucleophile (e.g. an alcohol) and a Lewis acid (e.g. a trialkyl borate) with heating to give the corresponding 2xe2x80x2-O-substituted pyrimidine nucleoside. In preferred embodiments of the present invention, a 2S,2xe2x80x2, or 2,2xe2x80x2-anhydropyrimidine nucleoside is treated with an alcohol of formula R1xe2x80x94OH in the presence of a weak nucleophile to give the respective 2xe2x80x2-Oxe2x80x94R1-pyrimidine nucleoside or 2S analog. The process is amenable to both small and large scale synthesis of 2xe2x80x2-O-substituted pyrimidine nucleosides.
2S,2xe2x80x2-Anhydropyrimidine nucleosides can be made from pyrimidine nucleosides following the procedure of, for example, Townsend, Leroy B., supra. See also Ueda, T., Tanaka, H., Chem. Pharm. Bull. (Toykyo), 1970, 18, 149. The 2S,2xe2x80x2-anhydropyrimidine nucleoside can then be treated with a weak nucleophile (e.g. an alcohol) and a Lewis acid (e.g. a trialkyl borate) according to the methods of the invention to give the corresponding 2xe2x80x2-O-substituted 2S-pyrimidine nucleoside.
2xe2x80x2-O-substituted pyrimidine nucleosides and 2xe2x80x2-O-substituted 2S-pyrimidine nucleosides can be converted into their respective DMT/amidites following standard methods and techniques to give the 1-[5-O-dimethoxytrityl-2-O-substituted-(3-Oxe2x80x94N,N-diisopropylamino-2-cyanoethylphosphite)]pyrimidine nucleoside or the 1-[5-O-dimethoxytrityl-2-O-substituted-(3-Oxe2x80x94N,N-diisopropylamino-2-cyanoethylphosphite)]-2S-pyrimidine nucleoside, each of which can be used as a monomeric subunit precursor in the synthesis of oligomeric compounds.
While we do not wish to be bound by a specific theory, it is thought that the mechanism of opening of the anhydropyrimidine nucleoside ring involves nucleophilic attack at the 2xe2x80x2-carbon by a weak nucleophile. It is thought that the nucleophilic attack is facilitated by the complexing of the Lewis acid to the bridging oxygen. The resulting complex is believed to activate the 2xe2x80x2-carbon to nucleophilic attack by the weak nucleophile. Another theory that may be applicable is that the Lewis acid complexes to the 5xe2x80x2 and 3xe2x80x2 hydroxyl groups, causing a conformational change in the molecule, enabling attack by the weak nucleophile to give the product.
In one embodiment of the invention the Lewis acid used is a trialkyl borate. The trialkyl borate can be purchased or prepared such that the borate alkyl groups are identical to the alkyl groups of the nucleophile. For example, when 2xe2x80x2-O-methyluridine is being prepared, trimethyl borate would preferably be the Lewis acid, and methanol preferably would be the nucleophile. Trimethyl, triethyl, and tripropyl borates are commercially available through Aldrich Chemical Company, Milwaukee, Wis.
Alternatively, trialkyl borates can be prepared from borane and the alcohol that corresponds to the desired substituent for the 2xe2x80x2-position as illustrated above. Typically, borane, which is available as a 1.0 M solution in THF, is reacted with 3 equivalents of the respective alcohol. When the evolution of hydrogen is completed the solution is concentrated to remove the THF. The resulting solution, which contains the desired trialkyl borane in the desired alcohol, can be used in the instant process to achieve opening of the anhydro ring as discussed above.
In one aspect of the present invention 2xe2x80x2-O-substituted pyrimidine nucleosides wherein the pyrimidine is a uridine or a substituted uridine can be converted (aminated) to the corresponding cytidine analog by known methods and techniques using, for example, 1,2,4-triazole. Typically, a 2xe2x80x2-O-substituted uridine nucleoside is first protected at the 3xe2x80x2-O and 5xe2x80x2-0 positions with a traditional protecting group such as, for example, acetic anhydride. 1,2,4-Triazole, in a traditional solvent (e.g. acetonitrile), is treated with POCl3 in the presence of a base (e.g. triethylamine) at low temperature. The protected 2xe2x80x2-O-substituted uridine nucleoside is dissolved in a traditional solvent and added to the solution containing triazole/POCl3. After sufficient time has passed, subsequent workup and purification yield the triazine-1-(3,5-di-O-protected-2-substituted)pyrimidine nucleoside. This compound can be converted into the cytosine analog by treatment with ammonia. The exocycloamino group can be protected, for example by treatment with benzoic anhydride, in a traditional solvent such as, for example, pyridine.
In a further aspect of the present invention 2xe2x80x2-O-substituted-2S-pyrimidine nucleosides wherein the pyrimidine is a uridine or a substituted uridine can be converted to the corresponding cytidine analog by known methods and techniques using 1,2,4-triazole. The procedure presented above is followed except that the 2S group is protected with an appropriate protecting group, e.g. toluoyl, prior to treatment with triazole.
The resulting 2xe2x80x2-O-substituted-uridine or 2xe2x80x2-O-substituted-2S-uridine can be converted into the respective DMT/amidite following standard methods and techniques to give 4-N-protected-1-(2-O-substituted-3-Oxe2x80x94N,N-diisopropyl-amino-2-cyanoethylphosphite-5-O-dimethoxytrityl)cytidine or the 2S analog. The 4-N-protected-1-(2-O-substituted-3-Oxe2x80x94N,N-diisopropylamino-2-cyanoethylphosphite-5-O-dimethoxytrityl)cytidine or the 2S analog can be used as a monomeric sub-unit precursor in the synthesis of oligomeric compounds.
In one aspect of the present invention, 2xe2x80x2-O-substituted-5-substituted uridine monomeric sub-units are prepared starting with 5-substituted uridine, synthesized as illustrated above. This compound is treated with dibutyltin oxide in an appropriate solvent, e.g. methanol, and purified. The resulting 1-(2xe2x80x2,3xe2x80x2,-di-O-butyltin-xcex2-D-erythro-pentofuranosyl)-5-substituted uridine is treated with a haloalkyl, a protected haloalkylamino, or a haloalkylimidazo compound (e.g. iodopropane) in an appropriate solvent to give the respective 2xe2x80x2-O-substituent. Aralkyl and aryl groups can be used in place of the alkyl group.
Pyrimidines bearing a variety of substitutions (i.e., pyrimidines bearing substituent groups) are known in the art. A representative list of such substituent groups amenable to the process of the present invention includes hydrocarbyl groups such as alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, carbocylo alkyl, carbocylo alkenyl, carbocylo aralkyl, aryl, aralkyl or substituted aralkyl. Preferably, the alkyl, alkenyl and alkynyl substituent groups have from 1 to about 30 carbons, with 1 to about 10 carbons being particularly preferred. Preferably, the aryl groups have from 6 to about 14 carbons, and aralkyl groups have from 7 to about 30 carbons. The substituent groups listed above can themselves bear substituent groups such as alkoxy, alcohol, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, or alkyl, aryl, alkenyl, or alkynyl groups, and ether groups.
Substitution of alkyl, alkenyl, and alkynyl groups at C-5 of uracil is reported in PCT application PCT/US92/10115, filed Nov. 24, 1992, and examples of alkyl substitutions are further disclosed by Manoharan, M., Antisense Research and Applications, Crooke and Lebleu, eds., CRC Press, Boca Raton, 1993. Further substitutions are reported in Townsend, L. B, ibid.
In the process of the present invention, a 2S, 2xe2x80x2 or 2,2xe2x80x2-anhydro pyrimidine nucleoside is treated with an alcohol of formula R1xe2x80x94OH. Representative R1 groups include alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, carbocylo alkyl, carbocylo alkenyl, carbocylo aralkyl, aryl, aralkyl or substituted aralkyl. Preferably, the alkyl, alkenyl and alkynyl R groups have from 1 to about 30 carbons, with 1 to about 10 carbons being particularly preferred. Preferably, aryl groups have from 6 to about 14 carbons, and aralkyl groups have from 7 to about 30 carbons. The R1 groups listed above can themselves bear substituent groups such as alkoxy, alcohol, amino, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, hydroxy, ether or further alkyl, aryl, alkenyl, or alkynyl groups.
2xe2x80x2-O-substituted pyrimidine nucleosides and 2xe2x80x2-O-substituted 2S-pyrimidine nucleosides (substituted pyrimidine nucleosides) prepared according to the process of the present invention can be used to prepare a variety of compounds including oligonucleotides, oligonucleosides, mixed oligo-nucleotides/nucleosides, and related compounds known in the art.
In the context of this invention, the term xe2x80x9coligonucleotidexe2x80x9d includes oligomers or polymers containing two or more nucleotide subunits. Nucleotides, as used herein, may include naturally occurring sugars, nucleobases, and intersugar (backbone) linkages as well as non-naturally occurring portions which function similarly. Such chemically modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases.
Specific examples of some preferred oligonucleotides envisioned for this invention may contain, in addition to phosphodiester intersugar linkages (backbones), modified intersugar linkages such as phosphorothioates, phosphotriesters, methyl phosphonates, chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
As used herein, the term oligonucleoside includes oligomers or polymers containing two or more nucleoside subunits having a non-phosphorous linking moiety. Oligonucleosides according to the invention have a ribofuranose moieties attached to a nucleobases through glycosyl bonds. An oligo-nucleotide/nucleoside for the purposes of the present invention is a mixed backbone oligomer having at least two nucleosides covalently bound by a non-phosphate linkage and at least one phosphorous containing covalent bond with a nucleotide, and wherein at least one of the monomeric nucleotide or nucleoside units is a 2xe2x80x2-O-substituted compound prepared using the process of the present invention. An oligo-nucleotide/nucleoside can additionally have a plurality of nucleotides and nucleosides coupled through phosphorous containing and/or non-phosphorous containing linkages.
Methods of coupling 2xe2x80x2-O-substituted compounds prepared using the process of the present invention include conversion to the phosphoramidite followed by solution phase or solid phase chemistries. Representative solution phase techniques are described in U.S. Pat. No. 5,210,264, issued May 11, 1993 and commonly assigned with this invention. Representative solid phase techniques are those typically employed for DNA and RNA synthesis utilizing standard phosphoramidite chemistry. (see, e.g., Protocols For Oligonucleotides And Analogs, Agrawal, S., ed., Humana Press, Totowa, N.J., 1993.) A preferred synthetic solid phase synthesis utilizes phosphoramidites as activated phosphates. The phosphoramidites utilize PIII chemistry. The intermediate phosphite compounds are subsequently oxidized to the pV state using known methods. This allows for synthesis of linkages including phosphodiester or phosphorothioate phosphate linkages depending upon oxidation conditions selected. Other phosphate linkages can also be generated. A representative list of suitable linkages includes phosphodiester, phosphotriester, hydrogen phosphonate, alkylphosphonate, alkylphosphonothioate, arylphosphonothioate, phosphorothioate, phosphorodithioate, phosphoramidate, ketone, sulfone, carbonate, thioamidate and other such moieties as are known in the art. Alkylphosphonothioate linkages are disclosed in WO 94/02499.
Standard methods and techniques used to increase the coupling efficiency of oligonucleotide synthesis include activation of 3xe2x80x2 and or 5xe2x80x2 functional groups. Some commonly activated groups are phosphate and phosphite which give the corresponding activated phosphate and activated phosphite (see e.g., Nucleic Acids in Chemistry and Biology; Blackburn, G. M., Gait M. J., Eds. Chemical Synthesis; IL: New York, 1990, Chapter 3, p. 98). Many others are known and can be used herein.
For the purposes of this specification, alkyl groups include but are not limited to substituted and unsubstituted straight chain, branch chain, and alicyclic hydrocarbons, including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl and other higher carbon alkyl groups. Further examples include 2-methyl-propyl, 2-methyl-4-ethylbutyl, 2,4-diethylpropyl, 3-propyl-butyl, 2,8-dibutyldecyl, 6,6-dimethyloctyl, 6-propyl-6-butyloctyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2-ethylhexyl and other branched chain groups.
Alkenyl groups useful in the invention include, but are not limited to, moieties derived from the above alkyl groups containing one or more carbonxe2x80x94carbon double bonds such as vinyl, allyl and crotyl.
Alkynyl groups useful in the invention include, but are not limited to, moieties derived from the above alkyl groups containing one or more carbonxe2x80x94carbon triple bonds such as propargyl.
The term aryl is intended to denote monocyclic and polycyclic aromatic groups including, for example, phenyl, naphthyl, xylyl, pyrrole, and furyl groups. Although aryl groups (e.g., imidazo groups) can include as few as 3 carbon atoms, preferred aryl groups have 6 to about 14 carbon atoms, more preferably 6 to about 10 carbon atoms.
The term aralkyl is intended to denote groups containing both alkyl and aryl portions wherein the point of attachment of such groups is through an alkyl portion thereof. Benzyl groups provide one example of an aralkyl group.
A number of substituent groups can be introduced into compounds of the invention in a protected (blocked) form and subsequently de-protected to form a final, desired compound. Substituent groups include groups covalently attached to the pyrimidine ring and the R1 group of the alcohol R1xe2x80x94OH described above. In general, protecting groups render chemical functionality inert to specific reaction conditions and can be appended to and removed from such functionality in a molecule without substantially damaging the remainder of the molecule. See, e.g., Greene and Wuts, Protective Groups in Organic Synthesis, 2d ed, John Wiley and Sons, New York, 1991. For example, amino groups can be protected as phthalimido groups or as 9-fluorenyl-methoxycarbonyl (FMOC) groups and carboxyl groups can be protected as fluorenylmethyl groups. Representative hydroxyl protecting groups are described by Beaucage, et al., Tetrahedron 1992, 48, 2223. Preferred hydroxyl protecting groups are acid-labile, such as the trityl, monomethoxytrityl, dimethoxytrityl, and trimethoxytrityl groups.
Solid supports according to the invention include controlled pore glass (CPG), oxalyl-controlled pore glass (see, e.g., Alul, et al., Nucleic Acids Research 1991, 19, 1527), TentaGel Supportxe2x80x94an aminopolyethyleneglycol derivatized support (see, e.g., Wright, et al., Tetrahedron Letters 1993, 34, 3373) or Porosxe2x80x94a copolymer of polystyrene/divinylbenzene. Many other solid supports are commercially available and amenable to the present invention.
An activated solid support in the context of the present invention is a solid support that has been derivatized with a functional group or treated with a reactive moiety such that the resulting activated solid support is chemically active towards reaction with a monomeric subunit or a nucleoside dimer of the invention.
In some preferred embodiments the processes of the invention are used to prepare 2xe2x80x2-O-substituted-5-halo pyrimidine nucleosides. 2xe2x80x2-O-substituted-5-halo uridine may be prepared from 5-F, xe2x80x94Cl, xe2x80x94Br, and xe2x80x94I uracils, which are available from Aldrich Chemical Company. The 2xe2x80x2-O-substituted 5-halo uridine can be converted into the cytidine analog as discussed below.
In the process of the invention, the alcohol, lewis acid and anhydropyrimidine nucleoside are treated in a reaction vessel. The reaction vessel may be any container known in the art to be suitable for reactions wherein reactants are heated. Preferably, the reaction vessel is a pressure sealed vessel, and more preferably, a pressure bomb.
In preferred embodiments the alcohol, lewis acid and anhydropyrimidine nucleoside in an appropriate solvent such as dioxane and are treated by heating at from about 120xc2x0 C. to about 200xc2x0 C. In especially preferred embodiments the alcohol, lewis acid, anhydropyrimidine nucleoside and solvent are heated in a pressure sealed vessel. A pressure bomb is particularly preferred.
The term Lewis acid, as used herein, has its usual meaning as a molecule or ion which can combine with another molecule or ion by forming a covalent bond with two electrons from the second molecule or ion. For use in the process of the invention, a Lewis acid is considered as an electron deficient species that can accept a pair of electrons. Particularly preferred are the di and tri valent hard acids. More particular are the divalent and trivalent cations of di and tri valent metals and their complexes including magnesium, calcium, aluminum, zinc, chromium, copper, boron, tin, mercury, iron, manganese, cadmium, gallium and barium. Their complex will include, but are not limited to, hydroxides, alkyls, alkoxides, di and trihalides particularly the trichlorides and trifluorides and organic acid ligands such as acetates. Especially preferred examples of Lewis acids useful in the instant process are borates, particularly alkyl borates.
Oligonucleotides including 2xe2x80x2-O-methylpyrimidine nucleotides have been evaluated in studies to assess their effectiveness in the inhibition of gene expression, as well as their hybridization affinity to complementary RNA. One such study (see Monia, B. P., et.al., J. Biol. Chem., 1993, 268, 14514-14522) using 2xe2x80x2-modified oligonucleotides containing 2xe2x80x2-deoxy gaps and uniformly modified 2xe2x80x2-modified oligonucleotides has shown that the 2xe2x80x2-modified oligonucleotides have greater affinity for complementary RNA than the corresponding unmodified 2xe2x80x2-deoxyoligonucleotides. The 2xe2x80x2-modified oligonucleotides containing 2xe2x80x2-deoxy gaps also showed activity against the expression of the Ha-ras oncogene in cells.
In another study which included uniformly modified 2xe2x80x2-O-substituted oligoribonucleotides, nuclease stabilities and melting temperatures (Tm) were compared for fully modified oligoribonucleotide sequences containing 2xe2x80x2-fluoro, 2xe2x80x2-O-methyl, 2xe2x80x2-O-propyl, and 2xe2x80x2-o-pentyl nucleotides. Modifications, with the exception of 2xe2x80x2-O-pentyl, were observed to increase the Tm of duplexes formed with complementary RNA. Modified homoduplexes showed significantly higher TmS, with the following Tm order: 2xe2x80x2-fluoro:2xe2x80x2-fluoro greater than 2xe2x80x2-o-propyl:2xe2x80x2-O-propyl  greater than 2xe2x80x2-O-methyl:2xe2x80x2-O-methyl greater than RNA:RNA greater than DNA:DNA. The nuclease stability of 2xe2x80x2-modified oligoribonucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. The stability imparted by 2xe2x80x2-modifications was observed to correlate with the size of the modification. An additional level of nuclease stability was present in oligoribonucleotides having the potential for forming secondary structure, but only for 2xe2x80x2 modified oligoribonucleotides and not for 2xe2x80x2-deoxy oligoribonucleotides.
Additional objects, advantages, and novel features of this invention will become apparent to those skilled in the art upon examination of the examples thereof provided below.